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A Nanopipette-based SERS Aptasensor for Subcellular Localization of Cancer Biomarker in Single Cells Sumaira Hanif, Hai-Ling Liu, Saud Asif Ahmed, Jin-Mei Yang, Yue Zhou, Jie Pang, Lina Ji, Xing-Hua Xia, and Kang Wang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b02147 • Publication Date (Web): 21 Aug 2017 Downloaded from http://pubs.acs.org on August 22, 2017
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Analytical Chemistry
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A Nanopipette-based SERS Aptasensor for Subcellular
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Localization of Cancer Biomarker in Single Cells
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Sumaira Hanifa, Hai-Ling Liua, Saud Asif Ahmeda, Jin-Mei Yanga, Yue Zhoua, Jie Panga, Li-Na
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Jib*, Xing-Hua Xiaa and Kang Wanga*
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a: State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and
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Chemical Engineering, Nanjing University, Nanjing 210023, China.
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b: State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing
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University, Nanjing 210023, China;
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*Corresponding author:
[email protected];
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Abstract: Single cell analysis is essential for understanding the heterogeneity, behaviors of
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cells, and diversity of target analyte in different subcellular regions. Nucleolin (NCL) is a
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multifunctional protein that is markedly overexpressed in most of the cancers cells. The variant
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expression levels of NCL in subcellular regions have marked influence on cancer proliferation
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and treatments. However, the specificity of available methods to identify the cancer biomarkers
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is limited because of the high level of subcellular matrix effect. Herein, we proposed a novel
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technique to increase both the molecular and spectral specificity of cancer diagnosis by using
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aptamers affinity based portable nanopipette with distinctive surface-enhanced Raman scattering
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(SERS) activities. The aptamers-functionalized gold-coated nanopipette was used to capture
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target, while p-mercaptobenzonitrile (MBN) and complementary DNA modified Ag
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nanoparticles (AgNPs) worked as Raman reporter to produce SERS signal. The SERS signal of
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Raman nanotag was lost upon NCL capturing via modified DNA aptamers on nanoprobe, which
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further helped to verify the specificity of nanoprobe. For proof of concept, NCL protein was
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specifically extracted from different cell lines by aptamers modified SERS active nanoprobe.
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The nanoprobes manifested specifically good affinity for NCL with a dissociation constant Kd of
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36 nM and provided a 1000-fold higher specificity against other competing proteins.
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Furthermore, the Raman reporter moiety has vibrational frequency in the spectroscopically silent
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region (1800-2300 cm-1) with a negligible matrix effect from cell analysis. The subcellular
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localization and spatial distribution of NCL were successfully achieved in various types of cells
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including MCF-7A, HeLa and MCF-10A cells. This type of probing technique for single cell
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analysis could lead to develop a new perspective in cancer diagnosis and treatment at cellular
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level.
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Keywords: Aptasensor, nanopipette, nucleolin, SERS signal on/off, single cells, spatial
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distribution
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Single cell analysis is essential for unveiling the heterogeneity, behaviors of cells, and diversity
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of target analyte localizations.1,2 The single cell heterogeneity dictates a multitude of functions
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that could be helpful to investigate the development of disease states (prognosis and proliferation)
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and get information of carcinogenic transformations.3-5 A number of different methods have been
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developed for single cells analysis, including capillary electrophoresis (CE),6,7 mass
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spectrometry,8, flow cytometry,9 and laser induced fluorescence (LIF)10,11 based assays. Besides
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these well-developed techniques, a huge space is available due to some unsolved problems in
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cancer diagnostics at cellular level.12-14
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Surface-enhanced Raman scattering (SERS) has emerged as an appealing potential
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detection tool for cell study due to its unprecedented optical properties.15 As well characterized
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SERS substrates, metal nanoparticles16-18 undergo indocile aggregation in cells19,20 and usually
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interferes with original SERS signal21. To overcome this issue, SERS substrates with fixed
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configuration have been used, such as carbon nanopipettes, optical fibers and SERS-active glass
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nanopipettes.
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biomarkers in the single living cells.23 However, to the best of our knowledge, biocompatible and
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portable aptamers based SERS nanoprobes have not been explored yet for subcellular
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localization of biological targets in single cells.
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Recently, SERS-active glass pipette has been successfully applied for probing
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As a multifunctional protein associated with numerous biomolecules (DNA, RNA, and
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other small molecules), nucleolin (NCL) plays an essential role in cell division.24 NCL is
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localized at various parts of the cells including nucleus, cytoplasm and cell membrane with the
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characteristic functionalities.25 More importantly, the altered expression levels of subcellular
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NCL are found to be closely related to various types of cancers,26 including colorectal cancer,27
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human breast and lung cancer cells.28 Usually, an immunofluorescence assay is used to examine
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the various localization of NCL in cells.29 However, immunofluorescence technique can hardly
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examine the spatial distribution of NCL at single cell level. Previously, a SERS-based analysis
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was reported via a DNA barcode-assay for NCL detection at the cells surface while its
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fabrication steps were complicated and it was not applicable for subcellular distribution.
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Therefore, it is highly desirable to develop an alternative and non-invasive method for detecting
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and mapping the expression of NCL at subcellular level.
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To detect and inhibit the proliferation of cancer cells, several efforts have been put to
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develop NCL binding molecules, such as aptamers (AS1411) and peptides.31-33 AS1411 is a 26-
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mer G-rich DNA Aptamer, which is highly stable against nucleases.34,35 Herein, we developed a
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simple SERS-based nanoprobe for subcellular localization of NCL in a single living cell via
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aptamers based affinity. SERS-active glass nanopipette was functionalized with AS1411 then
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hybridized with versatile Raman reporter and cDNA modified Ag nanotags for the recognization
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of NCL. The principle and procedure of aptamers affinity SERS assay are illustrated in Scheme 1.
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The thin layer of gold film coated on glass nanopipette with ~200 nm outer diameter was used as
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based substrate, while thiolated NCL aptamers were modified on the surface of Au-coated
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nanopipette. Meanwhile, Raman nanotags prepared by immobilizing thiolated complementary
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DNA (cDNA) of AS1411 and MBN as Raman reporter on 55±5 nm AgNPs were used for the
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detection. The developed SERS nanoprobe detection is relied on the SERS signal-off response of
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MBN on the exposure of target molecule due to release of Raman nanotags from hybridized
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SERS-active nanopipette. The bi-metallic nature of Au and Ag plasmon and MBN as Raman
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reporter with characteristic peak (2223 cm-1) in cell silent region provided a versatile detection
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platform for single cell analysis. For proof of concept, the SERS nanoprobe was precisely
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inserted into nucleus, cytoplasm and near cell surface in different living cell lines (MCF-7, MCF-
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10A and HeLa cells). The nanoprobe exhibits astonishing results for subcellular localized study
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and spatial distribution of biomarker in single cells. Thus, the burgeoning field of SERS active
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nanopipettes can be further explored by applying various aptamer-affinity approaches for single
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living cell study.
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EXPERIMENTAL SECTION
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Reagents and materials. Oligonucleotides used in the present study were purchased from
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Shanghai
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(China), and the sequences are given below:
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Nucleolin Aptamer (AS1411):
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5`-(CH2)6-S-S-(CH2)6-GGTGGTGGTGGTTGTGGTGGTGGTGG-3`
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Complementary ssDNA:
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3`-(CH2)6-S-S-(CH2)6-CCACCACCACCAACACCACCACCACC-5`
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4-mercaptobenzonitrile (MBN, 95%) was purchased from Nanjing Norris Pharm Technology Co.
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Ltd. Bovine serum albumin (BSA), horseradish peroxidase (HRP), β-Casein (β-Cas),
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cytochrome-c (Cyt-c), transferrin (Trf), TE buffer (100-X, pH 7.4) and nucleolin (NCL) from
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calf intestinal mucosa were all from Sigma-Aldrich (St. Louis, MO, USA). Hydrogen
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tetrachloroaurate trihydrate (HAuCl4 3H2O, 99.9%) was purchased from Alfa-Aesar (Shanghai
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China). Silver nitrate (AgNO3), Triton-X 100, trisodium citrate, NaOH, NaH2PO4, Na2HPO4,
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glucose, potassium bicarbonate (KHCO3), NaOH, HCl (36%), NaCl, KCl, MgCl2, CaCl2,
Sangon
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KH2PO4, K2HPO4, EDTA, glycerol and anhydrous ethanol of analytical grade were purchased
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from the Nanjing reagent company. All these reagents were used without further purification.
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Water was purified with a Milli-Q Advantage A10 (Millipore, Milford, MA, USA), and was used
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to prepare all solutions. Borosilicate glass capillaries with filament (0.58 mm I.D., 1.0 mm O.D.)
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were purchased from Sutter Instrument Co.
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Instrumentation. CO2-laser-based pipet puller (P-2000, Sutter Instrument Co.) was used to
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fabricate the glass nanopipettes. Scanning electron microscopic (SEM) characterization was
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carried out on a FE-SEM S-4800 system (Hitachi, Tokyo, Japan). Transmission electron
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microscopy (TEM) characterization was performed on a JEM-1011 system (JEOL, Tokyo,
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Japan). Raman and SERS experiments were conducted on a Renishaw InVia Reflex confocal
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microscope (Renishaw, UK) equipped with a high-resolution grating with 1800 grooves/cm,
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additional band-pass filter optics, and a CCD camera. All measurements were carried out using a
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He-Ne laser (λ = 633 nm; laser power at spot, 1 mW; excitation laser line: 1s integration time
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and 1 accumulation). The laser was focused onto the sample by using a ×50 objective lenses with
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(N.A. 0.75), providing a spatial resolution of 1 µm. Wavelength calibration was performed by
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measuring silicon wafers through a ×50 objective, assessing the first-order phonon band of
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silicon (Si) at 520 cm-1. The spectra were recorded using the Renishaw's WiRE (Windows-based
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Raman Environment) software and analyzed with Origin Pro 8.6 software. Each spectrum
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baseline was corrected except noise test. A three-dimensional manipulator equipped on an
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inverted microscope (Nikon Ti-E) was used to precisely insert extraction microprobes into single
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cells under investigation.
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Preparation of Raman nanotags
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Synthesis of silver nanoparticles. Silver nanoparticles (AgNPs) with a diameter of 55±5 nm
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were prepared according to a method by Lee and Meisel.36 In brief, AgNO3 (36 mg) was
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dissolved in 200 mL water and brought to boil under continuous stirring. Then, 4 mL of 1% (w/v)
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trisodium citrate was added. The mixture was boiled with stirring for about 1 h and then cooled
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down to room temperature naturally. The solution was stored at 4 °C in refrigerator for further
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use.
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Immobilization of cDNA and Raman reporter on AgNPs. The cDNA of NCL aptamers and
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Raman reporter was immobilized on AgNPs by using following procedure.26 10 µL of 100 µM
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cDNA of aptamer(3’-thiol) were added to freshly prepared AgNPs (1 mL), and incubated at 4 °C
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for 6 h. The obtained solution was centrifuged for 10 min at 10,000 rpm to remove the excess
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reagents, the precipitate was washed and centrifuged repeatedly for three times. The resulting
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cDNA-Ag nanoprobe was dispersed into 1 mL phosphate buffer solution (PBS, 0.01 M, pH 7.4).
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To modify Raman reporter, 1mL cDNA-AgNPs were incubated with 1 µL of MBN (1 mM
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dissolved in 0.2 M NaOH) for 1 h at room temperature under constant stirring. Then cDNA-
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AgNPs were washed with 0.01 M PBS (pH 7.4) three times by centrifugation and re-suspended
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in 1 mL PBS (pH 7.4) , stored at 4 °C for further use.
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Fabrication of SERS nanoprobe
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Fabrication of glass nanopipette. Borosilicate glass without filament (0.58 mm I.D., 1.0 mm
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O.D., Sutter Instrument Co.) was fabricated by a CO2-laser-based pipette puller (P-2000, Sutter
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Instrument Co.).
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Preparation of Au coated nanoprobe. For the preparation of Au-coated nanopipette, a gold
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layer was coated onto the surface of glass nanopipette by a previously reported chemo-deposition
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method.37 Briefly, the nanopipettes (200 nm O.D.) were immersed in a solution containing 12
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mM HAuCl4, 0.5 M KHCO3 and 25 mM glucose for 3-4 h at 45 °C until a clear gold layer
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formed on the surface of each nanopipette. After washing with water and anhydrous ethanol 2-3
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min for each, the Au-coated nanoprobes dried in air at room temperature.
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Immobilization of aptamers on Au-nanoprobe. The aptamers modification on nanoprobe was
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performed by immersing in 50 µL of 100 µM 5’-thiol aptamer in 0.01 M PBS and incubated at
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4 °C in dark for 5 h. Then, excess reagent was removed by washing with 0.01 M PBS (pH 7.4)
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for 2 min. The resulting aptamer-modified nanoprobes were stored in refrigerator at 4 °C for
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further use.
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NCL detection assay in solution
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Hybridization of Raman nanotag on SERS nanoprobe. Au nanoprobes modified with
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aptamers were immersed in 50 µL of Raman nanotag solution and incubated at room temperature
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for 1 h. Then non-hybridized Raman nanotags were removed by washing with PBS (pH 7.4) for
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2 min and air dried before Raman measurement.
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Hybridized SERS nanoprobe for NCL detection. NCL was dissolved in protein binding buffer
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(PBB) to at a concentration of 0.1 µM. PBB was prepared by adding 140 mM NaCl, 5 mM KCl,
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1 mM MgCl2 and 1 mM CaCl2 in 1× TE Buffer (pH 7). The as prepared SERS nanoprobe was
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immersed in NCL solution for 5 min at room temperature then washed with PBB and air dried
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before Raman analysis.
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Selectivity test. The selectivity of the SERS-nanoprobe was investigated against different
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proteins including BSA, Trf, HRP, Cyt-c and β-casein. The SERS nanoprobes were incubated in
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1 nM NCL or 1 µM interferants in PBB (pH 7) for 5 min each. Then nanoprobes were washed
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with PBB (pH 7) for 2 min, dried at room temperature and detected by the Raman.
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Cellular analysis
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Cell culture. NCL-positive MCF-7, normal MCF-10A, HeLa cells were purchased from the Cell
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Bank of Type Culture Collection of Chinese Academy of Sciences (Shanghai). The cells were
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cultured in DMEM (Gibco) media containing 10% fetal bovine serum (Gibco), 100 mg/mL
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streptomycin and 100 U/mL penicillin at 37 °C for 48 h under 5% CO2. Finally, cell culture
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media were removed and the cells lingered on the cell culture dishes were washed with 1× PBS
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buffer for three times then kept in 1× PBS buffer for further use.
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SERS nanoprobe for single cells analysis. With the help of an inverted microscope (Nikon Ti-
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E equipped with a three-dimensional manipulator), the nanoprobe was inserted into single cells
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anchored on the bottom of a petri dish. The nanoprobe was precisely localized at different
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regions (nucleus, cytoplasm and cell surface) of single cells under microscopy and kept still for 5
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min for each measurement. Meanwhile, the cells were kept in 1× PBS at room temperature for all
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measurements. After NCL extraction from each region of cells, the nanoprobes were washed
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with 1× PBS for 2 min to remove unwanted matrix from the nanoprobe, then dried at room
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temperature and measured for SERS signals. For spatial distribution study, the nanoprobe was
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kept horizontal when it approached the nucleus of single cells by passing the cytoplasmic region.
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Then the nanoprobe was washed with 1× PBS for 2 min after extraction, dried and measured for
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SERS signals. All the experiments were repeated in triplicates to confirm the results. Meanwhile,
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blank as negative control experiment was also conducted before each analysis.
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RESULTS AND DISCUSSION
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Characterization of the SERS nanoprobe and Raman nanotags. In this study, a smooth and
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uniform gold layer on was coated the surface of glass nanopipettes in order to generate
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plasmonic effect. The combination of bimetallic nanostructures (i.e. Au film and AgNPs) to
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generate an enhanced plasmon has gained more interest in SERS based detection materials
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because of its unique plasmon coupling effect and other physiochemical properties.38 With an
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outer diameter of ~250 nm and a taper angle of 33˚, the Au coated glass nanopipette is sharp
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enough to penetrate into the nucleus of a living cell. The SEM images of SERS nanoprobe before
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and after its hybridization with Raman nanotag are shown in Figure 1A and 1B. For the
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synthesis of Raman nanotags, AgNPs with an average diameter of 55±5 nm were prepared
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(Figure S1A). The AgNPs were modified with MBN as Raman reporter and thiolated aptamers
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complementary DNA as binding receptors for Raman nanotags. TEM images in Figure S1B and
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S1C showed that the AgNPs retained its shape and dispersity before and after the modification
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with MBN and cDNA. The localized surface plasmon resonance (LSPR) characterization of
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MBN and cDNA modified AgNPs showed no apparent shifts in the LSPR spectrum, suggesting
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that the modified AgNPs were well dispersed (Figure S2).
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The optical response of hybridized SERS nanoprobe against NCL is also presented by the
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SEM images in Figure S3. The SERS response of aptamers modified Au-coated nanoprobe is
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shown in Figure S4, which represents many characteristic peaks in DNA fingerprint region while
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no peaks at cell silent region (highlighted in grey). The detailed characteristic peaks with
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assigned vibrations are listed in Table S1. The sharp peak at 2223 cm-1 is assigned to
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characteristic vibration of cyanide ν(-C≡N) functional group of MBN, and is selected for further
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experimental characterizations. The MBN is a robust Raman reporter molecule for cell based
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study due to its simple structure and sharp Raman peak at cell silent region (~2223 cm-1).39,40
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The SERS response of Raman nanotags before and after modification is given in Figure S5,
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which indicates a sharp and clear peak at 2223 cm-1 while the other cDNA characteristic peak
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was overcome by MBN peaks in DNA fingerprint region.
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The incubation time and concentration of thiolated aptamers and cDNA were optimized
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before analysis. The 5’ labeled DNA sequences (MB labeled aptamers and alkyne labeled cDNA)
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were used to optimize DNA concentrations on the surface of Au coated glass nanopipette and
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AgNPs, respectively. The SERS response at peak 1623 cm-1 of MB labeled aptamers modified
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nanoprobe is shown in Figure S6A with other characteristic peaks given in Table S1. The
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optimized concentration and incubation time for aptamers modification on Au nanoprobe were
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100 µM and 5 h, respectively (Figures S6B). Similarly, the alkyne tagged cDNA gave a
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characteristic peak at 2010 cm-1 that was taken as a reference peak for optimization (Figure S7A).
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The optimized concentration and incubation time for cDNA modification on AgNPs were 100
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µM and 6h, respectively (Figures S7B). Meanwhile, the previously optimized concentration and
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the maximum incubation time of MBN modification on metal surface were 1 mM and 1 h to get
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a maximum saturation point.39 It is important to note that a consistent increase in SERS
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intensities is observed with gradually increasing incubation time for the DNA sequences
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modifications even for lower concentrations (10 and 50 µM for aptamers and cDNA). However,
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higher concentrations have been chosen in order to get maximum Raman signal of modified
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DNAs on the plasmonic surfaces of Au nanoprobe and Ag nanotag.
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NCL detection. The aptamer modified SERS nanoprobe was incubated with Raman nanotags to
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form hybridization complex between complementary DNA sequences. This interaction between
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Au surfaces of nanoprobe with MBN-attached Raman nanotags resulted in the amplification of
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SERS signal at peak position of 2223 cm-1. The amplified signal within the hot spots of Au
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nanoprobe and Raman nanotags was specifically lost if target protein NCL was present in the
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solution as shown in Figure 1C. The binding tendency of modified aptamers towards the target
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NCL was much higher as compared to complementary sequence hybridization.41 As shown in
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Figure S8, the complete SERS spectra including DNA and protein fingerprint regions manifest
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the alterations in characteristic peaks region of DNA after capturing protein. The reaction time of
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SERS nanoprobe for NCL detection was also optimized to be 5 min (Figure S9). Similar
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response of modified aptamers towards NCL was also observed in previous reports,42 indicating
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that the binding efficiency of SERS nanoprobe modified aptamers for the target molecule can be
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utilized for single cell analysis. Furthermore, SERS mapping image in Figure S10 indicates the
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even distribution of Raman nanotags after hybridization on SERS nanoprobe. Therefore the
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fabricated nanoprobe has the ability to provide quantitative information about the target analyte
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(NCL) within complex biological samples.
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Binding affinity of SERS nanoprobe. The binding affinity of SERS nanoprobe in vitro was
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determined by using increasing concentrations of NCL (0.5 to 500 nM). As shown in Figure 2A,
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the SERS signal of MBN gradually decreased with the increasing concentration of NCL. Figure
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2B showed that the decrease in SERS intensity at peak 2223 cm-1 became almost static when
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NCL concentration was above 100 nM, suggesting the NCL capturing by SERS nanoprobe might
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reach to saturation. The plot of SERS intensity at 2223 cm-1 against varying concentration of
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NCL showed a significant binding affinity with an apparent Kd of 36 nM (the detailed is shown
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in supporting information). The experimentally calculated limit of detection by using standard
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deviation formula (S/N>3) was 0.8 nM for in vitro NCL with a linear range of 1-50 nM (inset of
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Figure 2B), which was comparable to the previous report.42 Such a good binding affinity ensures
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the formation of a stable complex between aptamers and NCL with significant detection limit to
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release the hybridized Raman nanotags into the solution.43
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Selectivity of the SERS nanoprobe. The selectivity of SERS nanoprobe was checked against a
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variety of interfering proteins such as BSA, HRP, β-Cas, Cyt-C and Trf. As shown in Figure 3,
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the SERS nanoprobe exhibited excellent specificity against the interfering proteins because no
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SERS signal variation was observed for them as compared to blank. Even though the
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concentration of interfering proteins was 1000-fold higher than that of NCL (especially for β-Cas
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being phosphoprotein similar to NCL), the interfering proteins showed no or minimal SERS
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response. Thus, the SERS nanoprobes are highly selective for the target molecule due to
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aptamers based affinity.
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Reproducibility of SERS nanoprobe. The SERS signal reproducibility of the SERS nanoprobe
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was determined in terms of the coefficient of variation for hybridized nanoprobes before and
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after NCL capturing. The SERS responses of 10 different Au nanoprobes were measured for
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peak position at 2223 cm-1 before and after NCL detection (Figure S11). The relative standard
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deviation (RSD) value of 8.6% manifested the good reproducibility of fabricated SERS-based
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detection approach on Au coated glass nanopipette to work in sample with nanoliter volume.
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Subcellular localization and spatial distribution of NCL in single cells. To understand the
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subcellular detection and spatial distribution of NCL, SERS nanoprobe was precisely inserted
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within single cancer cells (MCF-7 and HeLa) and normal (MCF-10A) cells. For the subcellular
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localizations of NCL, different SERS nanoprobes were individually inserted in perpendicular
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way to the various cellular compartments as in nucleus, cytoplasm and kept near inner cell
293
membrane for 5 min at each region as shown in Figure 4A. The SERS spectra in Figure S12
294
depicted that the overexpressed NCL levels in cancer cells led to a tremendous decrease in SERS
295
signal of MBN at peak 2223 cm-1 in all regions including cytoplasm and cell surface. Meanwhile,
296
no significant decrease in SERS signal was observed in cytoplasmic and cell surface regions of
297
normal MCF-10A cells. Interestingly, due to the occurrence of SERS signal of MBN within
298
silent region of cell, negligible cell matrix effect and no SERS spectral overlapping were
299
observed as compared to blank. To further investigate the semi-quantitative analysis, percentage
300
loss in SERS spectra intensity was observed for NCL localization in different cell lines (Figure
301
4B). For instance, blank nanoprobes has 100% SERS signal and after NCL capturing the
302
intensity dropped to 11.2%, 12.5% and 12% in nucleus, cytoplasm and cell surface of MCF-7
303
cancer cell, respectively. For HeLa cells, the SERS intensity dropped to 11.8%, 34.6% and 47%
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for nucleus, cytoplasm and cell surface regions, respectively. Conversely, for normal cell (MCF-
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10A) the drop of SERS intensity was not consistent in all regions. The SERS intensity of MCF-
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10A dropped to 12.2% in nucleus, which is the native synthetic region for NCL in all normal
307
cells. The relatively higher percentage loss of SERS signal in cytoplasmic and cell surface
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regions of cancer cells also verified the overexpressed level of NCL in these regions.
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Furthermore, the expression levels of NCL in cancer and normal cells were also detected in
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cytoplasmic and nuclear extracts of these cell lines (Figure S13). Figure S14 showed a low
311
degree of relative SERS intensity variations among different cellular regions, indicating that
312
NCL was homogenously distributed in cellular extracts and excessive free NCL molecules were
313
available for capturing. All these results for the subcellular detection of NCL and variation
314
within different cancer lines are in well agreement with previously published reports.44 Besides,
315
the spatial distribution of NCL on single nanoprobe was also investigated by inserting the Au
316
nanoprobe horizontally passing by the cytoplasm towards the nucleus of cell. After extraction the
317
SERS spectra were collected by starting from nucleus (N) inserted region towards the cell
318
surface (S) region of the nanoprobes with the total available surface area of 7-8 µm for Raman
319
measurements, and the schematic presentation is given in Figure S15. SERS spectral response in
320
Figure 5A showed a uniform distribution of SERS signal of MBN at peak position 2223 cm-1
321
before extraction of any NCL from the cells. The corresponding SERS spectra in Figure 5B, 5C
322
and 5D showed that the NCL was not evenly distributed in MCF-7 MCF-10A and HeLa cells.
323
Furthermore, the spatial distribution of NCL in various cell lines was also monitored via SERS
324
mapping technique as shown in Figure S16. Therefore, we can extract molecular information
325
about heterogeneously overexpressed NCL levels among different types of cancer cell lines (as
326
HeLa and MCF-7 are used in current study) which is highly important in cancer diagnosis.45
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Hence, the SERS nanoprobes have the tendency to distinguish the subcellular NCL levels and
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also can be implied for study of spatial distribution of any cancer biomarker with high precision
329
in individual cells. Moreover, our proposed approach with some alteration (cDNA modified
330
nanopipette and hybridization with aptamers attached nanoparticles) could be further useful to
331
develop an extended drug delivery setup or cancer therapy platform for localized region in
332
cancer cells. Such a simple, specific and sensitive probing strategy of cancer cells will also be
333
helpful for accessing biomolecular information at cellular level by merging it to in-situ SERS
334
detection platform.
335 336
Conclusion
337
In conclusion, we proposed a novel design of the SERS nanoprobe for cancer biomarker
338
detection at a subcellular level. The fabricated SERS nanoprobe has provided several beneficial
339
properties. First, the Raman reporter labeled AgNPs can aggregate on Au nanotip, and provide
340
significantly enhanced and reproducible SERS signal, which is selectively lost in the presence of
341
target. Second, the characteristic peak of MBN at 2223 cm-1 in the Raman silent region (cell and
342
other biomolecules) enables to detect the distribution of biomarker in single cells and reflect the
343
physiological and pathological states of the cancer cells. Third, the recognition nanoprobe
344
contains aptamers that can be easily replaced by the other types of aptamers to develop diverse
345
ranges of detecting platforms for different biological targets. Lastly, the design of fabricated
346
detection nanoprobe is highly flexible to be converted into the cancer treatment therapy setup
347
with a little variation, as our future perspective. Particularly, the successful detection of NCL at
348
subcellular level along the spatial distribution for the first time may provide a potential approach
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for detection of other proteins at low expression level in a single living cell and opens a new
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horizon for cancer cell research.
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ASSOCIATED CONTENT
353
Supporting Information
354
The Supporting Information is available free of charge on the ACS Publications website.
355
Synthesis routes, additional optimization, additional characterization and additional validation
356
are provided in the supporting information.
357 358
AUTHOR INFORMATION
359
* Corresponding Author:
[email protected],
[email protected] 360
Tel.: +86-25-89687436
361 362
ACKNOWLEDGEMENTS
363
This work was supported by the grants from the National Natural Science Foundation of China
364
(21575059 and 21627806) and the National Science Fund for Creative Research Groups
365
(21121091).
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FIGURE CAPTIONS
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Scheme 1. Schematic illustration of aptamers affinity based SERS nanoprobe for probing NCL
437
in single cells.
438 439
Figure 1. SEM images of aptamers modified Au-nanoprobe before (A) and after hybridization
440
(B) with Raman nanotag. (C) SERS response of hybridized nanoprobe before and after nucleolin
441
(NCL) detection. SERS nanoprobe was incubated in 0.1 µM NCL solution for 5 min at room
442
temperature.
443 444
Figure 2. (A) Concentration-dependent SERS spectra of hybridized nanoprobe for NCL in PBB
445
(pH 7). (B) Plots of SERS intensity at 2223 cm-1 versus different concentrations of NCL. Binding
446
isotherm data was calculated with Hill fitting (R2 = 0.995). Inset graph indicates the linear fitting
447
for different NCL concentrations from 1 to 50 nM with value of R2 = 0.985. Error bars were
448
estimated from three repeated measurements.
449 450
Figure 3. Selectivity of SERS nanoprobe in the presence of different interferants. Bar chart
451
plotted for SERS intensity at 2223 cm-1 versus different proteins and blank (having no protein)
452
under similar conditions. Sample: 100 nM of target NCL protein and 100 µM of other competing
453
proteins dissolved in PBB (pH 7) while blank contain only buffer. Error bars were estimated
454
from three repeated measurements.
455 456
Figure 4. Optical images show the NCL extraction from (A) nucleus, (B) cytoplasm and (C) cell
457
surface of individual cell. The scale bar is 5 µm. (D) SERS intensities at 2223 cm-1 for each
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nanoprobe inserted into different cell lines (MCF-7, MCF-10A and HeLa) within different
459
regions of cells (nucleus, cytoplasm and cell surface).
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460 461
Figure 5. SERS spectra for spatial distribution of NCL probing in different cell lines. (A) Blank
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SERS nanoprobe (B) MCF-7 cells (C) MCF-10A cells and (D) HeLa cells. Raman analysis was
463
performed for 7-8 µm region of SERS nanoprobe with a roughly estimated step distance of 1 µm
464
for each measurement, while N and S referred as nucleus and surface regions of the cells,
465
respectively.
466
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Scheme 1
477
Scheme 1
478 479 480 481 482 483 484 485 486 487 488 489
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C
Aptamer modified SERS nanoprobe
1000 counts
Raman nanotags hybridized SERS nanoprobe Hybridized SERS nanoprobe after NCL detection
SERS intensity
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500 2000
2100
2200
2300
501
Raman Shift (cm )
502
Figure 1
-1
503 504 505 506 507 508 509 510 511 512
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515
0 nM 0.5 nM 1 nM 10 nM 25 nM 50 nM 100 nM 200 nM 300 nM 400 nM 500 nM
516 517 518
2000
2100
2200
2300
2400
520
6000 4000
Raman Shift (cm )
7695 6840 5985 5130 4275 3420 0
2000
10 20 30 40 50 Conentration of NCL (nM)
Hill Fitting
0 0
-1
519
Sample Blank without NCL
8000
SERS intensity at 2223 cm-1
A
514
B10000 1000 counts
SERS intensity at 2223 cm-1
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200
300
400
Concentration of NCL (nM)
Figure 2
521 522 523 524 525 526 527 528 529 530 531 532 533 534 535
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536 537 538 539 540 541 542
10000 SERS intensity at 2223 cm-1
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
8000 6000 4000 2000 0
NCL Blank BSA HRP β-Cas Cyt-C Trf
543 544
Figure 3
545 546 547 548 549 550 551 552 553 554 555 556 557 558
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D12000 SERS intensity at 2223 cm-1
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Nucleus Cytoplasm Cell Surface
10000 8000 6000 4000 2000 0
569 570
Blank
MCF-10A
MCF-7
Figure 4
571 572 573 574 575 576 577 578 579 580 29 ACS Paragon Plus Environment
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8000 6000
584
4000 2000
585
5000
B
SERS Intensity
10000
A
583
4000 3000 2000 1000 0
0
S
2000
2100
2200
2300
on / it i
N 2000
2400
2100
2200
2300
Po s
N
587
Po si tio n/ µm
586
µm
S
2400
Raman Shift (cm-1)
Raman Shift (cm-1)
10000
C
8000 6000
590
4000 2000
591
10000
D
SERS Intensity
589
8000 6000 4000 2000 0
0
S
n/ µ t io N
593 2000
594
2100
2200
2300
N 2000
2400
2100
2300
Raman Shift (cm-1)
Raman Shift (cm-1)
595 596
2200
Figure 5
597 598 599 600 601
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Po si tio n/
m
µm
S
592
SERS Intensity
588
SERS Intensity
582
Po si
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Table of Content
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603 604
An aptamer modified gold nanopipette was decorated by Raman reporter-covered AgNPs to
605
form a signal-off nanoprobe. The subcellular localization and spatial distribution of NCL were
606
probed in various types of cells
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